The present invention relates to new and existing indolo[2,1-b]quinazoline-6,12-dione derivatives which are useful in killing malaria parasites, antimalarial compositions containing the compounds, and to methods of treating malaria with the compounds and compositions, alone or in combination with other antimalarial agents both in vitro and in vivo.
The current global situation in respect to malaria has recently been detailed by Peters [W. Peters, xe2x80x9cDrug Resistance in Malaria Parasites of Animals and Manxe2x80x9d, Advances in Parasitology, vol. 41, pp 1-62 (1998)]. Currently, about two billion people are exposed to malaria and 400 million are infected with the disease. Between 100-200 million new cases occur each year. There are approximately 1-2 million deaths annually due to malaria. The global situation is worsening. The pertinent facts are these: very few new antimalarial drugs have been introduced in the past quarter century; there is massive pressure for the development of drug resistance due to the presence of large numbers of non-immune people in areas where malaria is efficiently transmitted; and resistance by Plasmodium falciparum and Plasmodium vivax to chloroquine is being documented in an increasingly wide geographic area.
Other frontline drugs currently used for the treatment and prevention of malaria such as mefloquine and halofantrine, are becoming increasingly ineffective. Newly introduced artemsinin analogs (artesunate and artemether), while effective for the treatment of malaria, may be too toxic for long term administration required for prophylaxis. As a result, the idea of malaria eradication has been abandoned and replaced with the more realistic target of malaria control.
Indolo[2,1-b]quinazoline-6,12-dione is a compound with a long history [see C. W. Bird, Tetrahedron, 19, 901 (1963), and references therein]. The structure of the compound has been verified by x-ray crystallography: M. Brufani, et al., Experientia, 27, 1249 (1971); W. Fedeli, et al., J. Chem. Soc. Perkin Transactions 2, 621 (1974). Early developments were described by Friedlander and Reschdestwensky [Ber., 48, 1843 (1915). Numerous synthetic approaches to the parent compound have been described: H. Karpf, et al., Tet. Let, 3007 (1978); L. A. Mitscher, et al., Heterocycles, 15, 1017 (1981); L. Capuano, et al., Chem. Ber., 116, 741 (1983); S. Euguchi, et al., 33, 153 (1992). Indolo[2,1-b]quinazoline-6,12-dione is also a naturally occurring compound, that is found in the higher plants such as Couroupita guianensis Aubl [Bergman, et al., Tet. Let., 2625 (1977)]; Strobilanthes cusia [G. Honda, et al., Planta Medica, 37, 172, (1979)]; Polygonum tinctorum and Isatis tinctorum [G. Honda, et al., Planta Medica, 38, 275 (1980). It is produced by Candida liplytica when grown in media containing an excess of tryptophan, hence its name, tryptanthrin. Indolo[2,1-b]quinzoline-6,12-dione has been shown to possess antibacterial activity against a variety of pathogenic bacteria, particularly the causative agent of tuberculosis, Mycobacterium tuberculosis. Antibacterial activity is also claimed against Staphylococcus aureus, Klebsiella pneumoniae, Mycobacterium smegmatis, and the fungi, Candida albicans [Mitscher, et al., xe2x80x9cAntimicrobial Agents from Higher Plants. New Synthesis and Bioactivity of Tryptanthrin (Indolo[2,1-b]quinazoline-6,12-dione) and its Analoguesxe2x80x9d, Heterocycles 15, 1017-1021 (1981); Honda, G. and Tabata., M., xe2x80x9cIsolation of Antifungal Principal Tryptanthrin from Strobilanthes Cusia O. Kuntze,xe2x80x9d, Planta Medica, J Med. Plant Res., 36, 85-86 (1979); Mitscher, et al., xe2x80x9cPart I. Antitubercular Agents from Higher Plants: Synthesis and In Vitro Activity of Indolo[2,1-b]quinazoline-6,12-diones and Related Analogsxe2x80x9d, Abstracts of Papers, 35 International Congress of Antimicrobial Agents and Chemotherapy, Abstract F 16, San Diego, Calif., 1995; Baker, W. xe2x80x9cPart II. Antitubercular Agents from Higher Plants: Antimycobacterial Activity of Azaindoloquinazolines. Novel Agents against Sensitive and Multi-drug Resistant Tuberculosisxe2x80x9d, Abstracts of Papers, 35 International Congress of Antimicrobial Agents and Chemotherapy, Abstract F17, San Diego, Calif., 1995. To date, however, there has been no evidence or indication that Indolo[2,1-b]quinazoline-6,12-dione and derivatives exhibit anti-malarial activity against malaria parasites or would be useful in treating malaria in vivo or in vitro.
Historically, the first antimalarial drugs stemmed from natural remedies. The quinchona alkaloids were utilized for centuries before their active principals, alkaloids such as quinine and quinidine, were isolated and shown to be effective in themselves against malaria. These compounds are devoid of useful clinical antibacterial activity. The discovery of the first synthetic antimalarial drugs was prompted by the selective staining of plasmodium tissues by vital stains. This lead, based upon the organic chemistry of synthetic dyes, led after a period of perhaps twenty years, to the discovery of two classes of quinoline antimalarial drugs, the 4-aminoquinolines (such as chloroquine) and the 8-aminoquinolines (such as primaquine). The biochemical basis of the antimalarial action of these agents, despite investigations spanning the last 50 years, is still unknown. Notwithstanding their extensive use as antimalarial agents, these compounds have found no clinical utility against bacterial species.
Some antibacterial agents have found application in the therapy and prevention of malaria. These include compounds whose mechanisms of antibacterial action are well documented. Those interfering with folate metabolism are the best known. These include the drug combination pyrimethamine-sulfadoxine, and dapsone. However, well known antimalarials which inhibit the metabolism of folate within the plasmodium, such as proguanil and cycloguanil, have not found application as antibacterial agents despite their extensive clinical application as antimalarial drugs. Doxycycline is used for malaria prophylaxis, and recently azithromycin C has shown antimalarial activity. Many extremely powerful antibacterial agents, such as the penicillins and cephalosporins, are devoid of antimalarial activity.
U.S. Pat. No. 5,441,955, the disclosure of which is expressly incorporated herein by reference, describes the general Formula I described herein, with the exception that it does not disclose that X can be a side chain necessary to make the compound of Formula I a prodrug. However, the ""955 patent focuses on antibacterial compounds for treating bacterial infections, but fails to contemplate treating malaria parasites with these compounds or using them as antimalarial agents. Thus, the inventors of this invention have discovered the unexpected result of these particular compounds, and the compounds with side chains rendering the compounds prodrugs, as antimalarial agents.
That there is little relationship between antibacterial activity in a drug such as described in the ""955 patent and antimalarial activity in a drug is not surprising. The bacteria and the plasmodia are very distant genetically: Bacteria are prokaryotes and plasmodia are eukaryotes. Thus, the search for acceptable antimalarial drugs is more difficult than the search for antibacterials as the metabolic processes of the plasmodia more closely resemble those of their eukaryotic hosts while the genetic and metabolic gap between bacterial and mammals is large as they belong to different kingdoms. Hence, an agent that is useful for treating a bacterial infection is not necessarily useful for treating a parasitic infection like malaria.
The inventors of the present invention have surprisingly discovered that Indolo[2,1-b]quinazoline-6,12-dione, and substituted derivatives, exhibit potent in vitro antimalarial activity against Plasmodium falciparum. Highly active compounds show IC50 values (50% inhibitory concentration) in the 0.43 to 10 ng/mL concentration range, about one one-thousandth of the concentrations necessary to inhibit bacteria. Furthermore, these compounds are also highly active against strains of Plasmodium falciparum which are up to 5000-fold resistant to atovoquone, 50-fold resistant to chloroquine, and 20-fold resistant to mefloquine. Therefore, this invention provides methods for inhibiting the growth of malaria parasites in vitro and provides methods for the prevention and treatment of malaria in vivo using indolo[2,1-b]quinazoline-6,12-dione compounds of formula I. 
wherein A, B, C, D, E, F, G and H are independently selected from carbon and nitrogen, or A and B or C and D can be taken together to be nitrogen or sulfur, with the proviso that not more than three of A, B, C, D, E, F, G and H are other than carbon; wherein R1 through R8 are independently selected from the group consisting of, but not limited to, the halogens (F, Cl, Br, and I), alkyl groups, trifluoromethyl groups, methoxyl groups, the carboxy methyl or carboxy ethyl group (COOCH3 or COOCH2CH3), nitro, aryl, heteroaryl, cyano, amino, dialkylaminoalkyl, 1-(4-alkylpiperazinyl), and the pharmaceutically acceptable salts thereof; and wherein X is independently selected from the group consisting of any atom especially oxygen, or any side chain necessary to make the indolo[2,1-b]quinazoline-6,12-dione compound a xe2x80x9cprodrugxe2x80x9d as the term is understood by one of ordinary skill in the art of medicinal chemistry. In other words, a side chain having a structure where a carbon-nitrogen double bond bears substituents that make the prodrug more water soluble and bioavailable. Improved bioavailability results in a lower effective dosage. It also makes more predictable and uniform absorbance which increases the predictability of the response which in turn increases safety by reducing drug side effects.
An example of a prodrug containing a labile carbon-nitrogen double bond side chain exocyclic to the tryptanthin structure is shown in Formula II. Such an example may be more water soluble, but can be readily converted to the tryptantrin structure through hydrolysis of the carbon-nitrogen bond to give tryptanthrin. 
Another example of a labile structure attached to the keto position which can increase the aqueous solubility of the tryptanthrin nucleous, but will be hydrolysed back to tryptanthrin in the blood stream is shown in Formula III. 
These examples of prodrugs are not intended to limit the invention in any way. Other side chains are contemplated that would make the indolo[2,1-b]quinazoline-6,12-dione compound of the above formula I a prodrug as the term is understood by one of ordinary skill in the art of medicinal chemistry. Prodrugs are described in Medicimal Chemestry Principles and Practice, Frank D. Cane, Royal Society of Chemistry, Cambridge England, Chapter 14, pp215-218 (1994), which is herein incorporated by reference.